Cell-free platform for the functional characterization of intracellular ion transporters

Abstract

148 pagesIn biotechnology, the study of membrane proteins is of high interest due to their unique physical properties used as invaluable tools for the development of new therapeutics. However, honing down on the structural and functional properties of membrane proteins is no easy feat, owing to challenges in expression, purification, and the availability of high-throughput functional readout methods. Historically, cell-based protein production methods have dominated the field of membrane protein studies; but now, cell-free protein synthesis (CFPS) approaches are beginning to both complement and rival traditional expression methods. Regarding readout methods, maintaining the structural and functional properties of membrane proteins requires an easily controllable and specific lipid environment that promotes lateral diffusion and protein-lipid interactions. Here, I present a customizable cell-free platform for the functional assembly and integration of membrane proteins in both liposomes and supported lipid bilayers (SLBs) to qualitatively assess membrane protein activity. Our platform merges the convenience and scalability of CFPS with two functional readout methods: a fluorescence assay in liposomes and an SLB bioelectronic platform. With these tools, we can directly detect the activity of difficult-to-study membrane proteins like intracellular ion transporters. Additionally, we can further characterize membrane protein expression, orientation, and function in an isolated and controllable environment. With the momentum of developing improved CFPS and biosensing technologies, this platform can aid in extending the limits of membrane protein biotechnology